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The endoplasmic reticulum-associated degradation of the epithelial sodium channel requires a unique complement of molecular chaperones.

Buck TM, Kolb AR, Boyd CR, Kleyman TR, Brodsky JL - Mol. Biol. Cell (2010)

Bottom Line: Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD).We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells.Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

ABSTRACT
The epithelial sodium channel (ENaC) is composed of a single copy of an alpha-, beta-, and gamma-subunit and plays an essential role in water and salt balance. Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD). To define how the ENaC subunits are selected for degradation, we developed novel yeast expression systems for each ENaC subunit. Data from this analysis suggested that ENaC subunits display folding defects in more than one compartment and that subunit turnover might require a unique group of factors. Consistent with this hypothesis, yeast lacking the lumenal Hsp40s, Jem1 and Scj1, exhibited defects in ENaC degradation, whereas BiP function was dispensable. We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells. Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

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The ERAD of ENaC subunits is dependent on both the Hrd1 and Doa10 ubiquitin ligases. Cycloheximide chase reactions were performed as described in Materials and Methods in HRD1/DOA10 (●), hrd1Δ (▴), doa10Δ (▵), or hrd1Δdoa10Δ (○) yeast strains (Pagant et al., 2007) expressing C-terminally HA-tagged α-, β-, or γ-ENaC. Chase reactions were performed at 37°C, lysates were immunoblotted with anti-HA (ENaC) or with anti-Sec61 (as a loading control). Data represent the means of 4–6 experiments, ±SEM.
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Figure 2: The ERAD of ENaC subunits is dependent on both the Hrd1 and Doa10 ubiquitin ligases. Cycloheximide chase reactions were performed as described in Materials and Methods in HRD1/DOA10 (●), hrd1Δ (▴), doa10Δ (▵), or hrd1Δdoa10Δ (○) yeast strains (Pagant et al., 2007) expressing C-terminally HA-tagged α-, β-, or γ-ENaC. Chase reactions were performed at 37°C, lysates were immunoblotted with anti-HA (ENaC) or with anti-Sec61 (as a loading control). Data represent the means of 4–6 experiments, ±SEM.

Mentions: In a recent report, we obtained preliminary data using the yeast system to establish that the turnover of a doubly tagged version of α-ENaC was proteasome-dependent, as observed in higher cell types (Kashlan et al., 2007). Degradation was also slowed in yeast containing a loss-of-function allele in UFD1, which encodes a member of the Cdc48 complex that extracts ubiquitinated substrates from the ER (Jentsch and Rumpf, 2007). Therefore, to determine which E3 ligase ubiquitinates the α-subunit, the protein was expressed in yeast lacking Hrd1, Doa10, or both Hrd1 and Doa10, and cycloheximide chase analyses were performed. Interestingly, we found that the degree of subunit stabilization was similar regardless of which mutant strain was used (Figure 2). We also developed expression systems for the β- and γ-subunits, and like the α-subunit expression system the proteins contained C-terminal HA tags. We found that the β- and γ-subunits were stabilized to a somewhat greater degree in the hrd1Δdoa10Δ mutant strain, but as observed with the α-subunit the degradation of the β- and γ-subunits was again similar in either the hrd1Δ or doa10Δ mutant.


The endoplasmic reticulum-associated degradation of the epithelial sodium channel requires a unique complement of molecular chaperones.

Buck TM, Kolb AR, Boyd CR, Kleyman TR, Brodsky JL - Mol. Biol. Cell (2010)

The ERAD of ENaC subunits is dependent on both the Hrd1 and Doa10 ubiquitin ligases. Cycloheximide chase reactions were performed as described in Materials and Methods in HRD1/DOA10 (●), hrd1Δ (▴), doa10Δ (▵), or hrd1Δdoa10Δ (○) yeast strains (Pagant et al., 2007) expressing C-terminally HA-tagged α-, β-, or γ-ENaC. Chase reactions were performed at 37°C, lysates were immunoblotted with anti-HA (ENaC) or with anti-Sec61 (as a loading control). Data represent the means of 4–6 experiments, ±SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2836957&req=5

Figure 2: The ERAD of ENaC subunits is dependent on both the Hrd1 and Doa10 ubiquitin ligases. Cycloheximide chase reactions were performed as described in Materials and Methods in HRD1/DOA10 (●), hrd1Δ (▴), doa10Δ (▵), or hrd1Δdoa10Δ (○) yeast strains (Pagant et al., 2007) expressing C-terminally HA-tagged α-, β-, or γ-ENaC. Chase reactions were performed at 37°C, lysates were immunoblotted with anti-HA (ENaC) or with anti-Sec61 (as a loading control). Data represent the means of 4–6 experiments, ±SEM.
Mentions: In a recent report, we obtained preliminary data using the yeast system to establish that the turnover of a doubly tagged version of α-ENaC was proteasome-dependent, as observed in higher cell types (Kashlan et al., 2007). Degradation was also slowed in yeast containing a loss-of-function allele in UFD1, which encodes a member of the Cdc48 complex that extracts ubiquitinated substrates from the ER (Jentsch and Rumpf, 2007). Therefore, to determine which E3 ligase ubiquitinates the α-subunit, the protein was expressed in yeast lacking Hrd1, Doa10, or both Hrd1 and Doa10, and cycloheximide chase analyses were performed. Interestingly, we found that the degree of subunit stabilization was similar regardless of which mutant strain was used (Figure 2). We also developed expression systems for the β- and γ-subunits, and like the α-subunit expression system the proteins contained C-terminal HA tags. We found that the β- and γ-subunits were stabilized to a somewhat greater degree in the hrd1Δdoa10Δ mutant strain, but as observed with the α-subunit the degradation of the β- and γ-subunits was again similar in either the hrd1Δ or doa10Δ mutant.

Bottom Line: Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD).We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells.Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

ABSTRACT
The epithelial sodium channel (ENaC) is composed of a single copy of an alpha-, beta-, and gamma-subunit and plays an essential role in water and salt balance. Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD). To define how the ENaC subunits are selected for degradation, we developed novel yeast expression systems for each ENaC subunit. Data from this analysis suggested that ENaC subunits display folding defects in more than one compartment and that subunit turnover might require a unique group of factors. Consistent with this hypothesis, yeast lacking the lumenal Hsp40s, Jem1 and Scj1, exhibited defects in ENaC degradation, whereas BiP function was dispensable. We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells. Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

Show MeSH
Related in: MedlinePlus